Materials and methods for detection and quantification of beta-n-methylamino-l-alanine

ABSTRACT

Provided herein are isotopically labeled reagents, including isotopically labeled small molecules and peptides, that can be used to detect and/or quantify β-N-methylamino-L-alanine (BMAA) in a sample. The reagents can be used as stable isotope labeled standards in analytical methods, including in conjunction with mass spectrometry, to detect and/or quantify BMAA in a sample, such as a protein sample from a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/368,437, filed Jul. 29, 2016, and U.S.Provisional Patent Application Ser. No. 62/368,562, filed Jul. 29, 2016,both of which are expressly incorporated herein by reference.

BACKGROUND

The non-protein amino acid β-N-Methylamino-L-Alanine (BMAA) isbiosynthetically produced in cyanobacteria. Human exposure to thisunnatural amino acid has been linked to neurological disorders,including amyotrophic lateral sclerosis (ALS) and parkinsonism dementiacomplex (PDC) like symptoms. Dietary exposure of BMAA in primates hasshown neurofibrillary tangles (NFT) and β-amyloid plaques, hallmarksigns of neuropathological disease. Additionally, cell culture studieshave shown that exogenous exposure to BMAA can result in incorporationof this non-protein amino acid in place of L-serine. Supplementaltreatment with L-serine has been shown to reduce the rate of BMAAincorporation and regression of neuropathological symptoms.

While these findings suggest that BMAA can be incorporated intoproteins, efforts to further study the role of BMAA in biologicalsystems has been hampered by the limited availability of suitableanalytical probes and methods. Improved analytical tools and methods areneeded to fully understand the qualitative and quantitative nature ofincorporation of BMAA in proteins, particularly human proteins, as wellas the mechanism and functional consequences of this process.

SUMMARY

Provided herein are isotopically labeled reagents, includingisotopically labeled small molecules and peptides, that can be used todetect and/or quantify β-N-methylamino-L-alanine (BMAA) in a sample. Thereagents can be used as stable isotope labeled standards in analyticalmethods, including in conjunction with mass spectrometry, to detectand/or quantify BMAA in a sample, such as a protein sample from asubject.

For example, provided herein are compositions that comprise anisotopically labeled compound defined by the formula below

where R represents hydrogen or an amine protecting group, and at easttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope (¹³C, ¹⁵N, and/or ¹⁸O). Incertain cases, R is hydrogen. In other cases, R represents an amineprotecting group, such as a 9-fluorenylmethyloxycarbonyl group. Theisotopically labeled compound can comprise at least 0.5% by weight ofthe composition.

In some embodiments, at least three (e.g., at least four, at least five,or at least six) of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and^(h)O are isotopically labeled with a stable isotope. In some cases, atleast two (e.g., at least three, or at least four) of ^(a)C, ^(b)C,^(c)C, ^(d)C, ^(e)N, and ^(f)N are isotopically labeled with a stableisotope. In certain cases, all of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and^(f)N are isotopically labeled with a stable isotope.

In some embodiments, the isotopically labeled compound can be defined bythe formula below

where R represents hydrogen or an amine protecting group (e.g., a9-fluorenylmethyloxycarbonyl group); the ¹³C isotopic enrichment factorfor ^(a)C, ^(b)c, ^(c)C, and ^(d)C is at least 25; and the ¹⁵N isotopicenrichment factor for ^(e)N and ^(f)N is at least 100. In certainembodiments, the ¹³C isotopic enrichment factor for ^(a)C, ^(b)C, ^(c)C,and ^(d)C can be at least 80 and the ¹⁵N isotopic enrichment factor for^(e)N and ^(f)N can be at least 200.

Compositions comprising a polypeptide that includes one or moreisotopically labeled β-N-methylamino-L-alanine (BMAA) residues are alsoprovided herein. The isotopically labeled polypeptide can comprise atleast 0.5% by weight of the composition. Each of the one or moreisotopically labeled BMAA residues can be isotopically labeled with oneor more (e.g., two or more) stable isotopes. For example, each of theone or more isotopically labeled BMAA residues can be defined by theformula below

where the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andthe ¹⁵N isotopic enrichment factor for ^(f)N is at least 100. In somecases, the ¹³C isotopic enrichment factor for ^(d)C is at least 80 andthe ¹⁵N isotopic enrichment factor for ^(f)N is at least 200. In somecases, the ¹³C isotopic enrichment factor for ^(a)C, ^(c)C, and ^(c)Ccan be at least 25 (e.g., at least 80). In some cases, the ¹⁵N isotopicenrichment factor for ^(e)N can be at least 100 (e.g., at least 200). Insome embodiments, the polypeptide can include a single isotopicallylabeled β-N-methylamino-L-alanine (BMAA) residue. In other embodiments,the polypeptide can include two or more isotopically labeled BMAAresidues (e.g., three or more isotopically labeled BMAA residues, fouror more isotopically labeled BMAA residues, five or more isotopicallylabeled BMAA residues, or ten or more isotopically labeled BMAAresidues).

In some embodiments, the polypeptide can be defined by the formula below

where m is an integer from 0 to 300 and n is an integer from 0 to 300,with the proviso that at least one of m and n is not 0; the ¹³C isotopicenrichment factor for ^(d)C is at least 25; the ¹⁵N isotopic enrichmentfactor for ^(f)N is at least 100; and independently for each occurrencein the polypeptide, R₁ is H and R₂ is selected from one of the following

or R₁ and R₂, together with the atoms to which they are attached, form afive-membered heterocycle defined by the structure below

In some embodiments, the ¹³C isotopic enrichment factor for ^(d)C is atleast 80 and the ¹⁵N isotopic enrichment factor for ^(f)N is at least200. In some embodiments, the sum of m and n is from 1 to 50 (e.g., from1 to 30). In some embodiments, the ¹³C isotopic enrichment factor for^(a)C, ^(b)C, and ^(c)C is at least 25 (e.g., at least 80). In someembodiments, the ¹⁵N isotopic enrichment factor for ^(e)N is at least100 (e.g., at least 200).

The isotopically labeled reagents and compositions described herein canbe used in a variety of analytical methods to detect and/or quantifyBMAA, such as to detect and/or quantify BMAA in a biological sample suchas a protein sample. For example, provided herein are methods forquantifying the amount of BMAA in a sample that comprise: (i) analyzinga test sample by mass spectrometry; (ii) spiking the test sample with adefined amount of an isotopically labeled compound defined by theformula below

where R represents hydrogen or an amine protecting group, and at leastthree of ^(a)C, ^(b)c, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope; to provide a BMAA-spikedsample; (iii) analyzing the BMAA-spiked sample by mass spectrometry; and(iv) determining the amount of BMAA in the test sample by isotopedilution analysis. Methods can further comprise preparing the testsample, the BMAA-spiked sample, or a combination thereof for analysis bymass spectrometry. For example, the test sample, the BMAA-spiked sample,or a combination thereof can be prepared for analysis by massspectrometry by a method comprising chemical reactions with flightenhancers, chemical fragmentation, enzymatic digestion, purification, ora combination thereof.

Also provided are methods for incorporating an isotopically-labeledβ-N-methylamino-L-alanine (BMAA) residue into a protein or peptide.Methods can comprise providing a protein or peptide that includes aphosphoserine residue; reacting the protein or peptide to convert thephosphoserine residue to an α,β-unsaturated amino acid residue; andreacting the α,β-unsaturated amino acid residue with methylamine toprovide a BMAA residue, wherein the methylamine is isotopically enrichedwith one or more stable isotopes. The methylamine can have a ¹³Cisotopic enrichment factor of at least 25 (e.g., at least 80) and an ¹⁵Nisotopic enrichment factor of at least 100 (e.g., at least 200).

DESCRIPTION OF DRAWINGS

FIG. 1 is and illustration of an example procedure used to convert aphosphoserine-containing peptide to BMAA-containing peptide. The peptidesequence shown in FIG. 1 is unique to superoxide dismustase 1 (SOD1).

FIGS. 2A and 2B illustrate the intact mass of stable isotope-labeled(SIL) phosphopeptide substrate and BMAA peptide product.

FIG. 3 illustrates the tandem mass spectrum of the phosphorylatedserine-containing stable isotope-labeled peptide.

FIG. 4 illustrates the tandem mass spectrum of a BMAA-containing stableisotope-labeled peptide, confirming localization of modification.

FIG. 5 demonstrates that co-eluting fragments confirm identity of theBMAA-containing peptide BMAA- and phosphoserine-containing peptides havedistinct retention times. The BMAA-containing peptide appears to havetwo peaks which suggests L and D isomers of the BMAA peptide have beenproduced.

FIG. 6 illustrates the conserved peak area of fragments specific to SILBMAA peptide that allow for confident identification of endogenouspeptide.

FIG. 7 is a plot showing that the elution time of SIL BMAA peptide isnecessary for correct identification of endogenous peptide. Withoutthis, the incorrect peak which is also within 5 ppm mass accuracy of theBMAA peptide of interest might be selected.

FIG. 8 demonstrates the ability of mass spectrometry to accuratelyidentify endogenous BMAA-containing peptide in SOD1 protein digestobtained from ALS erythrocytes.

FIG. 9 is a plot illustrating the co-elution of endogenous and stableisotope-labeled BMAA containing peptides. Co-elution aids in confidentidentification.

FIG. 10 illustrates the tandem mass spectrum of BMAA containing stableisotope-label (¹³C₄ ¹⁵N₂ β-N-Methylamino-L-Alanine).

FIG. 11 illustrates the tandem mass spectrum of a BMAA-containing stableisotope-labeled peptide. Fragment ions corresponding to the endogenouspeptide with BMAA were found at the expected location, confirminglocalization of modification.

FIG. 12 shows the occupancy or abundance of BMAA normalized to the total(phosphorylated+BMAA) after chemical conversion of serine to BMAA on 3peptides, across 5 potential reaction sites on Beta Casein.

FIG. 13 shows an extracted electropherogram of SIL BMAA and endogenousBMAA from ZipChip CZE-MS of Hydrolyzed b-casein after chemicalconversion of phosphoserine to BMAA.

DETAILED DESCRIPTION

Provided herein are isotopically labeled reagents, includingisotopically labeled small molecules and peptides, that can be used todetect and/or quantify β-N-methylamino-L-alanine (BMAA) in a sample. Thereagents can be used as stable isotope labeled standards in analyticalmethods, including in conjunction with mass spectrometry, to detectand/or quantify BMAA in a sample, such as a protein sample from asubject. The reagents can also be used as stable isotope labeledstandards in analytical methods, including in conjunction with massspectrometry, to detect and/or quantify free BMAA in, for example, anenvironmental sample.

Compositions

Provided herein are compositions that comprise an isotopically labeledcompound defined by the formula below

where R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope (i.e., ¹³C, ¹⁵N, and/or ¹⁸O).

In some embodiments, at least three (e.g., at least four, at least five,at least six, at least seven, or all eight) of ^(a)C, ^(b)C, ^(c)C,^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O are isotopically labeled with astable isotope. In certain embodiments, at least two (e.g., at leastthree, at least four, at least five, or all six) of ^(a)C, ^(b)C, ^(c)C,^(d)C, ^(e)N, and ^(f)N are isotopically labeled with a stable isotope.In particular embodiments, all of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, and^(f)N are isotopically labeled with a stable isotope.

In some embodiments, the isotopically labeled compound can be defined bythe formula below

where R represents hydrogen or an amine protecting group; the ¹³Cisotopic enrichment factor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is atleast 25; and the ¹⁵N isotopic enrichment factor for ^(e)N and ^(f)N isat least 100.

The term “isotopic enrichment factor,” as used herein, refers to theratio between the isotopic abundance (e.g., ¹³C, ¹⁵N, or ¹⁸O) at aspecified position in a compound and the naturally occurring abundanceof that isotope. The naturally occurring abundance of ¹³C is 1.1%. Thenaturally occurring abundance of ¹⁵N is 0.37%. The naturally occurringabundance of ¹⁸O is 0.204%.

In some embodiments, the ¹³C isotopic enrichment factor for ^(a)C,^(b)C, ^(c)C, and ^(d)C is at least 25 (27.5% ¹³C incorporation at eachposition), at least 30 (33% ¹³C incorporation at each position), atleast 35 (38.5% ¹³C incorporation at each position), at least 40 (44%¹³C incorporation at each position), at least 45 (49.5% ¹³Cincorporation at each position), at least 50 (55% ¹³C incorporation ateach position), at least 55 (60.5% ¹³C incorporation at each position),at least 60 (66% ¹³C incorporation at each position), at least 65 (71.5%¹³C incorporation at each position), at least 70 (77% ¹³C incorporationat each position), at least 75 (82.5% ¹³C incorporation at eachposition), at least 80 (88% ¹³C incorporation at each position), atleast 85 (93.5% ¹³C incorporation at each position), or at least 90 (99%¹³C incorporation at each position).

In some embodiments, the ¹⁵N isotopic enrichment factor for ^(e)N and^(f)N is at least 100 (37% ¹⁵N incorporation at each position), at least110 (40.7% ¹⁵N incorporation at each position), at least 120 (44.4% ¹⁵Nincorporation at each position), at least 130 (48.1% ¹⁵N incorporationat each position), at least 140 (51.8% ¹⁵N incorporation at eachposition), at least 150 (55.5% ¹⁵N incorporation at each position), atleast 160 (59.2% ¹⁵N incorporation at each position), at least 170(62.9% ¹⁵N incorporation at each position), at least 180 (66.6% ¹⁵Nincorporation at each position), at least 190 (70.3% ¹⁵N incorporationat each position), at least 200 (74% ¹⁵N incorporation at eachposition), at least 210 (77.7% ¹⁵N incorporation at each position), atleast 220 (81.4% ¹⁵N incorporation at each position), at least 230(85.1% ¹⁵N incorporation at each position), at least 240 (88.8% ¹⁵Nincorporation at each position), at least 250 (92.5% ¹⁵N incorporationat each position), at least 260 (96.2% ¹⁵N incorporation at eachposition), or at least 265 (98.05% ¹⁵N incorporation at each position).

In certain cases, R is hydrogen. In other embodiments, R represents anamine protecting group. Amine protecting groups are well known in theart, and include, for example, carbobenzyloxy (CBZ) groups,p-methoxybenzyl carbonyl (Moz) groups, tert-butyloxycarbonyl (BOC)groups, 9-fluorenylmethyloxycarbonyl (FMOC) groups, benzoyl (Bz) groups,benzyl (Bn) groups, carbamate groups, and p-methoxybenzyl (PMB) groups.In certain embodiments, R represents an amine protecting groupcompatible with solid phase peptide synthesis, such as a9-fluorenylmethyloxycarbonyl (FMOC) group or a tert-butyloxycarbonyl(BOC) group.

The composition can be, for example, a solution of the isotopicallylabeled compound in a solvent. Non-limiting examples of solvents includealiphatic solvents (e.g., pentane, hexanes, cyclohexane); aromaticand/or alkylated aromatic solvents such as benzene, toluene, xylene;hydrocarbon solvents; dichloromethane, chloroform, alcohols (e.g.,methanol, ethanol, isopropanol); esters (e.g., ethyl acetate); ketones(e.g., acetone); diethyl ether; dioxane; glycol ethers and glycol etheresters; tetrahydrofuran, dimethylformamide; acetonitrile; dimethylsulfoxide; water, saline, aqueous buffers (e.g., PBS buffer), andcombinations thereof. In certain examples, the composition can comprisean aqueous solution of the compound.

In some embodiments, the isotopically labeled compound can comprise atleast 0.5% by weight (e.g., at least 1% by weight, at least 1.5% byweight, at least 2% by weight, at least 2.5% by weight, at least 3% byweight, at least 3.5% by weight, at least 4% by weight, at least 4.5% byweight, or at least 1% by weight of the composition.

The isotopically labeled compounds described above can be prepared usingmethods known in the art. Representative methodologies for thepreparation of certain active agents are described below. Theappropriate route for synthesis of a given compound agent can beselected in view of the structure of the compound as a whole as itrelates to compatibility of functional groups, protecting groupstrategies, and the presence of labile bonds. In addition to thesynthetic methodologies discussed below, alternative reactions andstrategies useful for the preparation of the compounds disclosed hereinare known in the art. See, for example, March, “Advanced OrganicChemistry,” 5^(th) Edition, 2001, Wiley-Interscience Publication, NewYork).

Isotopically labeled amino acids, such as ¹³C/¹⁵N-labeled asparagine,are commercially available, and can serve as convenient startingmaterials for the isotopically labeled compounds described herein.Scheme 1 below illustrates an example method for the preparation of BMAAfrom asparagine. Compounds having a desired isotopic labeling (e.g.,incorporating stable isotopes at particular positions within thecompound) can be prepared by selecting reagents that include stableisotope labels at the appropriate positions with their framework (e.g.,¹³C/¹⁵N-labeled asparagine).

Also provided are compositions that include one or more isotopicallylabeled polypeptides. For example, compositions comprising a polypeptidethat includes one or more isotopically labeled β-N-methylamino-L-alanine(BMAA) residues are provided herein. The isotopically labeledpolypeptide can comprise at least 0.5% by weight of the composition.Each of the one or more isotopically labeled BMAA residues can beisotopically labeled with one or more (e.g., two or more) stableisotopes. For example, each of the one or more isotopically labeled BMAAresidues can be defined by the formula below

where the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andthe ¹⁵N isotopic enrichment factor for ^(f)N is at least 100.

In some embodiments, the ¹³C isotopic enrichment factor for ^(d)C is atleast 25 (27.5% ¹³C incorporation at each position), at least 30 (33%¹³C incorporation at each position), at least 35 (38.5% ¹³Cincorporation at each position), at least 40 (44% ¹³C incorporation ateach position), at least 45 (49.5% ¹³C incorporation at each position),at least 50 (55% ¹³C incorporation at each position), at least 55 (60.5%¹³C incorporation at each position), at least 60 (66% ¹³C incorporationat each position), at least 65 (71.5% ¹³C incorporation at eachposition), at least 70 (77% ¹³C incorporation at each position), atleast 75 (82.5% ¹³C incorporation at each position), at least 80 (88%¹³C incorporation at each position), at least 85 (93.5% ¹³Cincorporation at each position), or at least 90 (99% ¹³C incorporationat each position).

In some embodiments, the ¹⁵N isotopic enrichment factor for ^(f)N is atleast 100 (37% ¹⁵N incorporation at each position), at least 110 (40.7%¹⁵N incorporation at each position), at least 120 (44.4% ¹⁵Nincorporation at each position), at least 130 (48.1% ¹⁵N incorporationat each position), at least 140 (51.8% ¹⁵N incorporation at eachposition), at least 150 (55.5% ¹⁵N incorporation at each position), atleast 160 (59.2% ¹⁵N incorporation at each position), at least 170(62.9% ¹⁵N incorporation at each position), at least 180 (66.6% ¹⁵Nincorporation at each position), at least 190 (70.3% ¹⁵N incorporationat each position), at least 200 (74% ¹⁵N incorporation at eachposition), at least 210 (77.7% ¹⁵N incorporation at each position), atleast 220 (81.4% ¹⁵N incorporation at each position), at least 230(85.1% ¹⁵N incorporation at each position), at least 240 (88.8% ¹⁵Nincorporation at each position), at least 250 (92.5% ¹⁵N incorporationat each position), at least 260 (96.2% ¹⁵N incorporation at eachposition), or at least 265 (98.05% ¹⁵N incorporation at each position).

Optionally, ^(a)C, ^(b)C, and/or ^(c)C can also be labeled with a stableisotope. In some cases, the ¹³C isotopic enrichment factor for ^(a)C,^(b)C, and ^(c)C can be at least 25 (27.5% ¹³C incorporation at eachposition), at least 30 (33% ¹³C incorporation at each position), atleast 35 (38.5% ¹³C incorporation at each position), at least 40 (44%¹³C incorporation at each position), at least 45 (49.5% ¹³Cincorporation at each position), at least 50 (55% ¹³C incorporation ateach position), at least 55 (60.5% ¹³C incorporation at each position),at least 60 (66% ¹³C incorporation at each position), at least 65 (71.5%¹³C incorporation at each position), at least 70 (77% ¹³C incorporationat each position), at least 75 (82.5% ¹³C incorporation at eachposition), at least 80 (88% ¹³C incorporation at each position), atleast 85 (93.5% ¹³C incorporation at each position), or at least 90 (99%¹³C incorporation at each position).

Optionally, ^(e)N can also be labeled with a stable isotope. In somecases, the ¹⁵N isotopic enrichment factor for ^(e)N is at least 100 (37%¹⁵N incorporation at each position), at least 110 (40.7% ¹⁵Nincorporation at each position), at least 120 (44.4% ¹⁵N incorporationat each position), at least 130 (48.1% ¹⁵N incorporation at eachposition), at least 140 (51.8% ¹⁵N incorporation at each position), atleast 150 (55.5% ¹⁵N incorporation at each position), at least 160(59.2% ¹⁵N incorporation at each position), at least 170 (62.9% ¹⁵Nincorporation at each position), at least 180 (66.6% ¹⁵N incorporationat each position), at least 190 (70.3% ¹⁵N incorporation at eachposition), at least 200 (74% ¹⁵N incorporation at each position), atleast 210 (77.7% ¹⁵N incorporation at each position), at least 220(81.4% ¹⁵N incorporation at each position), at least 230 (85.1% ¹⁵Nincorporation at each position), at least 240 (88.8% ¹⁵N incorporationat each position), at least 250 (92.5% ¹⁵N incorporation at eachposition), at least 260 (96.2% ¹⁵N incorporation at each position), orat least 265 (98.05% ¹⁵N incorporation at each position).

In some embodiments, the polypeptide can include a single isotopicallylabeled BMAA residue. In other embodiments, the polypeptide can includetwo or more isotopically labeled BMAA residues (e.g., three or moreisotopically labeled BMAA residues, four or more isotopically labeledBMAA residues, five or more isotopically labeled BMAA residues, or tenor more isotopically labeled BMAA residues).

In some embodiments, the polypeptide can be defined by the formula below

where m is an integer from 0 to 300 and n is an integer from 0 to 300;the ¹³C isotopic enrichment factor for ^(d)C is at least 25; the ¹⁵Nisotopic enrichment factor for ^(f)N is at least 100; and independentlyfor each occurrence in the polypeptide, R₁ is H and R₂ is selected fromone of the following

or R₁ and R₂, together with the atoms to which they are attached, form afive-membered heterocycle defined by the structure below

In some embodiments, the polypeptide can be defined by the formula below

where m is an integer from 0 to 300 and n is an integer from 0 to 300,with the proviso that at least one of m and n is not 0; the ¹³C isotopicenrichment factor for ^(d)C is at least 25; the ¹⁵N isotopic enrichmentfactor for ^(f)N is at least 100; and independently for each occurrencein the polypeptide, R₁ is H and R₂ is selected from one of the following

or R₁ and R₂, together with the atoms to which they are attached, form afive-membered heterocycle defined by the structure below

In some embodiments, the ¹³C isotopic enrichment factor for ^(d)C is atleast 25 (27.5% ¹³C incorporation at each position), at least 30 (33%¹³C incorporation at each position), at least 35 (38.5% ¹³Cincorporation at each position), at least 40 (44% ¹³C incorporation ateach position), at least 45 (49.5% ¹³C incorporation at each position),at least 50 (55% ¹³C incorporation at each position), at least 55 (60.5%¹³C incorporation at each position), at least 60 (66% ¹³C incorporationat each position), at least 65 (71.5% ¹³C incorporation at eachposition), at least 70 (77% ¹³C incorporation at each position), atleast 75 (82.5% ¹³C incorporation at each position), at least 80 (88%¹³C incorporation at each position), at least 85 (93.5% ¹³Cincorporation at each position), or at least 90 (99% ¹³C incorporationat each position).

In some embodiments, the ¹⁵N isotopic enrichment factor for ^(f)N is atleast 100 (37% ¹⁵N incorporation at each position), at least 110 (40.7%¹⁵N incorporation at each position), at least 120 (44.4% ¹⁵Nincorporation at each position), at least 130 (48.1% ¹⁵N incorporationat each position), at least 140 (51.8% ¹⁵N incorporation at eachposition), at least 150 (55.5% ¹⁵N incorporation at each position), atleast 160 (59.2% ¹⁵N incorporation at each position), at least 170(62.9% ¹⁵N incorporation at each position), at least 180 (66.6% ¹⁵Nincorporation at each position), at least 190 (70.3% ¹⁵N incorporationat each position), at least 200 (74% ¹⁵N incorporation at eachposition), at least 210 (77.7% ¹⁵N incorporation at each position), atleast 220 (81.4% ¹⁵N incorporation at each position), at least 230(85.1% ¹⁵N incorporation at each position), at least 240 (88.8% ¹⁵Nincorporation at each position), at least 250 (92.5% ¹⁵N incorporationat each position), at least 260 (96.2% ¹⁵N incorporation at eachposition), or at least 265 (98.05% ¹⁵N incorporation at each position).

Optionally, ^(a)C, ^(b)C, and/or ^(c)C can also be labeled with a stableisotope. In some cases, the ¹³C isotopic enrichment factor for ^(a)C,^(b)C, and ^(c)C can be at least 25 (27.5% ¹³C incorporation at eachposition), at least 30 (33% ¹³C incorporation at each position), atleast 35 (38.5% ¹³C incorporation at each position), at least 40 (44%¹³C incorporation at each position), at least 45 (49.5% ¹³Cincorporation at each position), at least 50 (55% ¹³C incorporation ateach position), at least 55 (60.5% ¹³C incorporation at each position),at least 60 (66% ¹³C incorporation at each position), at least 65 (71.5%¹³C incorporation at each position), at least 70 (77% ¹³C incorporationat each position), at least 75 (82.5% ¹³C incorporation at eachposition), at least 80 (88% ¹³C incorporation at each position), atleast 85 (93.5% ¹³C incorporation at each position), or at least 90 (99%¹³C incorporation at each position).

Optionally, ^(e)N can also be labeled with a stable isotope. In somecases, the ¹⁵N isotopic enrichment factor for ^(e)N is at least 100 (37%¹⁵N incorporation at each position), at least 110 (40.7% ¹⁵Nincorporation at each position), at least 120 (44.4% ¹⁵N incorporationat each position), at least 130 (48.1% ¹⁵N incorporation at eachposition), at least 140 (51.8% ¹⁵N incorporation at each position), atleast 150 (55.5% ¹⁵N incorporation at each position), at least 160(59.2% ¹⁵N incorporation at each position), at least 170 (62.9% ¹⁵Nincorporation at each position), at least 180 (66.6% ¹⁵N incorporationat each position), at least 190 (70.3% ¹⁵N incorporation at eachposition), at least 200 (74% ¹⁵N incorporation at each position), atleast 210 (77.7% ¹⁵N incorporation at each position), at least 220(81.4% ¹⁵N incorporation at each position), at least 230 (85.1% ¹⁵Nincorporation at each position), at least 240 (88.8% ¹⁵N incorporationat each position), at least 250 (92.5% ¹⁵N incorporation at eachposition), at least 260 (96.2% ¹⁵N incorporation at each position), orat least 265 (98.05% ¹⁵N incorporation at each position).

In some embodiments, m can be at least 1 (e.g., at least 5, at least 10,at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 110, at least120, at least 130, at least 140, at least 150, at least 160, at least170, at least 180, at least 190, at least 200, at least 210, at least220, at least 230, at least 240, at least 250, at least 260, at least270, at least 280, or at least 290). In some embodiments, m can be 300or less (e.g., 290 or less, 280 or less, 270 or less, 260 or less, 250or less, 240 or less, 230 or less, 220 or less, 210 or less, 200 orless, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less,140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 orless, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 orless, 20 or less, 10 or less, or 5 or less). m can be an integer rangingfrom any of the minimum values described above to any of the maximumvalues described above. For example, in some embodiments, m can be aninteger from 1 to 300 (e.g., from 1 to 150, from 1 to 100, from 1 to 50,from 1 to 30, or from 1 to 10).

In some embodiments, n can be at least 1 (e.g., at least 5, at least 10,at least 20, at least 30, at least 40, at least 50, at least 60, atleast 70, at least 80, at least 90, at least 100, at least 110, at least120, at least 130, at least 140, at least 150, at least 160, at least170, at least 180, at least 190, at least 200, at least 210, at least220, at least 230, at least 240, at least 250, at least 260, at least270, at least 280, or at least 290). In some embodiments, n can be 300or less (e.g., 290 or less, 280 or less, 270 or less, 260 or less, 250or less, 240 or less, 230 or less, 220 or less, 210 or less, 200 orless, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less,140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 orless, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 orless, 20 or less, 10 or less, or 5 or less). n can be an integer rangingfrom any of the minimum values described above to any of the maximumvalues described above. For example, in some embodiments, n can be aninteger from 1 to 300 (e.g., from 1 to 150, from 1 to 100, from 1 to 50,from 1 to 30, or from 1 to 10).

In some embodiments, the sum of m and n can be at least 1 (e.g., atleast 5, at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 110, at least 120, at least 130, at least 140, at least 150, atleast 160, at least 170, at least 180, at least 190, at least 200, atleast 210, at least 220, at least 230, at least 240, at least 250, atleast 260, at least 270, at least 280, at least 290, at least 300, atleast 310, at least 320, at least 330, at least 340, at least 350, atleast 360, at least 370, at least 380, at least 390, at least 400, atleast 410, at least 420, at least 430, at least 440, at least 450, atleast 460, at least 470, at least 480, at least 490, at least 500, atleast 510, at least 520, at least 530, at least 540, at least 550, atleast 560, at least 570, at least 580, or at least 590). In someembodiments, the sum of m and n can be 600 or less (e.g., 590 or less,580 or less, 570 or less, 560 or less, 550 or less, 540 or less, 530 orless, 520 or less, 510 or less, 500 or less, 490 or less, 480 or less,470 or less, 460 or less, 450 or less, 440 or less, 430 or less, 420 orless, 410 or less, 400 or less, 390 or less, 380 or less, 370 or less,360 or less, 350 or less, 340 or less, 330 or less, 320 or less, 310 orless, 300 or less, 290 or less, 280 or less, 270 or less, 260 or less,250 or less, 240 or less, 230 or less, 220 or less, 210 or less, 200 orless, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less,140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 orless, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 orless, 20 or less, 10 or less, or 5 or less). The sum of m and n can sorange from any of the minimum values described above to any of themaximum values described above. For example, in some embodiments, thesum of m and n can be from 1 to 600 (e.g., from 1 to 300, from 1 to 150,from 1 to 100, from 1 to 50, from 1 to 30, from 1 to 10, from 5 to 300,from 5 to 150, from 5 to 100, from 5 to 50, from 5 to 30, or from 5 to10).

The composition can be, for example, a solution of the isotopicallylabeled peptide in a solvent. Non-limiting examples of solvents includealcohols (e.g., methanol, ethanol, isopropanol); esters (e.g., ethylacetate); ketones (e.g., acetone); diethyl ether; dioxane; glycol ethersand glycol ether esters; tetrahydrofuran, dimethylformamide;acetonitrile; dimethyl sulfoxide; water, saline, aqueous buffers (e.g.,PBS buffer), and combinations thereof. In certain examples, thecomposition can comprise an aqueous solution of the peptide.

In some embodiments, the isotopically labeled peptide can comprise atleast 0.5% by weight (e.g., at least 1% by weight, at least 1.5% byweight, at least 2% by weight, at least 2.5% by weight, at least 3% byweight, at least 3.5% by weight, at least 4% by weight, at least 4.5% byweight, or at least 1% by weight of the composition.

Also provided are compositions comprising an isotopically labeledpolypeptide that includes one or more β-N-methylamino-L-alanine (BMAA)residues (e.g., one or more BMAA residues that are not isotopicallylabeled) and one or more additional amino acid residues that are labeledwith one or more stable isotopes (¹³C, ¹⁵N, and/or ¹⁸O). In someembodiments, each residue labeled with one or more stable isotopes inthe polypeptide includes at least two (e.g., at least four, at leastfive, at least six, or more) stable isotopes. For example, at least two(e.g., at least four, at least five, at least six, or more) of thecarbon, nitrogen, and/or oxygen atoms in the residue can be isotopicallylabeled with a stable isotope. In some cases, at least two (e.g., atleast four, at least five, at least six, or more) of the carbon and/ornitrogen atoms in the residue can be isotopically labeled with a stableisotope. The isotopically labeled polypeptide can comprise at least 0.5%by weight of the composition.

In some embodiments, the polypeptide can include a single isotopicallylabeled residue. In other embodiments, the polypeptide can include twoor more isotopically labeled residues (e.g., three or more isotopicallylabeled residues, four or more isotopically labeled residues, five ormore isotopically labeled residues, or ten or more isotopically labeledresidues). In some embodiments, the polypeptide can include a singleBMAA residue. In other embodiments, the polypeptide can include two ormore BMAA residues (e.g., three or more BMAA residues, four or more BMAAresidues, five or more BMAA residues, or ten or more BMAA residues).

In certain embodiments, the isotopically labeled polypeptide can be apeptide that includes one or more BMAA residues and a terminal aminoacid residue (e.g., a terminal arginine residue) labeled with one ormore stable isotopes (¹³C, ¹⁵N, and/or ¹⁸O).

The composition can be, for example, a solution of the isotopicallylabeled peptide in a solvent. Non-limiting examples of solvents includealcohols (e.g., methanol, ethanol, isopropanol); esters (e.g., ethylacetate); ketones (e.g., acetone); diethyl ether; dioxane; glycol ethersand glycol ether esters; tetrahydrofuran; dimethylformamide;acetonitrile; dimethyl sulfoxide; water, saline, aqueous buffers (e.g.,PBS buffer), and combinations thereof. In certain examples, thecomposition can comprise an aqueous solution of the peptide.

In some embodiments, the isotopically labeled peptide can comprise atleast 0.5% by weight (e.g., at least 1% by weight, at least 1.5% byweight, at least 2% by weight, at least 2.5% by weight, at least 3% byweight, at least 3.5% by weight, at least 4% by weight, at least 4.5% byweight, or at least 1% by weight of the composition.

The peptides described above can be above can be prepared using avariety of methods known in the art. For example, peptides can beprepared using the isotopically labeled compounds described herein viasolid phase peptide synthesis. The proteins and peptides described abovecan also be prepared by chemical derivatization of one or more residueswithin the protein and peptide. For example, a protein or peptide havinga isotopically labeled β-N-methylamino-L-alanine (BMAA) residue can beprepared from a protein or peptide that includes a phosphoserineresidue. The protein or peptide can be reacted to convert thephosphoserine residue to an α,β-unsaturated amino acid residue. Onceactivated, the α,β-unsaturated amino acid residue can be reacted withmethylamine (e.g., methylamine that is isotopically enriched with one ormore stable isotopes), which undergoes a Michael-type addition to affordan isotopically labeled BMAA residue. This can involve, for example,reaction of the protein or peptide with methylamine-HCl (e.g., 1.0 M¹³C/¹⁵N-labeled methylamine) and Ba(OH)₂ (e.g., 0.1 M Ba(OH)₂) inwater/DMSO/EtOH (2:2:1) at basic pH (pH 12.5) and elevated temperature(e.g., 37° C.). Once complete, the reaction can be quenched with acid(e.g., acetic acid).

Full length natural or stable isotope labeled proteins comprising stableisotope labeled BMAA residues (e.g., incorporated at one or morespecific sites within the protein) can be prepared by first preparing aphosphoserine-containing protein using an amber stop codon and a tRNAsynthetase engineered to incorporate phosphoserine within the desiredprotein. See, for example, Rogerson, D. T. et al. “Efficient geneticencoding of phosphoserine and its nonhydrolyzable analog.” Nat. Chem.Biol., 2015, 7: 496-503. The protein can then be isolated, and thephosphoserine can be chemically converted into BMAA using the strategydescribed above.

Methods

The compounds, peptides, proteins, and compositions described herein canbe used detect and/or quantify BMAA in a sample (e.g., an environmentalsample or a biological sample), to accurately monitor BMAA exposure, todirect therapies, and in clinical diagnosis and prognosis. Thesematerials can be utilized in a wide range of analytical pipelines andapplications which require stable isotope labeled standards (SIL) forBMAA, protein specific peptides containing BMAA, and target proteinscontaining BMAA.

For example, the isotopically labeled reagents and compositionsdescribed herein can be used in a variety of analytical methods todetect and/or quantify BMAA, such as to detect and/or quantify BMAA in abiological sample such as a protein sample. The isotopically labeledcompounds described herein can be used to quantify free BMAA (e.g.,quantify free BMAA in an environmental sample or biological sample),quantify total levels of BMAA in a protein sample, and/or to quantifyprotein-specific BMAA incorporation (e.g., by upstream purification ofthe protein of interest prior to analysis). The isotopically labeledcompounds described herein can also be utilized in a stable isotopelabeling by amino acids in cell culture (SILAC) alone or in combinationwith other stable isotope labeled amino acids. The isotopically labeledcompounds described herein provide many analytical advantages overpotential alternatives, such as deuterated BMAA, which does not co-eluteand can undergo hydrogen-deuterium exchange in-solution or in the gasphase, significantly impacting identification and quantitation.

In some embodiments, methods for quantifying the amount of BMAA in asample can comprise: (i) analyzing a test sample by mass spectrometry;(ii) spiking the test sample with a defined amount of an isotopicallylabeled compound defined by the formula below

where R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope; to provide a BMAA-spikedsample; (iii) analyzing the BMAA-spiked sample by mass spectrometry; and(iv) determining the amount of BMAA in the test sample by isotopedilution analysis. Methods can further comprise preparing the testsample, the BMAA-spiked sample, or a combination thereof for analysis bymass spectrometry. For example, the test sample, the BMAA-spiked sample,or a combination thereof can be prepared for analysis by massspectrometry by a method comprising chemical reactions with flightenhancers, chemical fragmentation, enzymatic digestion, purification, ora combination thereof. In some embodiments, the isotopically labeledcompounds described herein can be used to detect and quantify BMAAobtained from the hydrolytic cleavage of amino acids from a targetprotein as well as for identification and quantification of BMAAincorporated into proteins at the peptide and protein level utilizingisotope dilution mass spectrometry.

The proteins and peptides described herein can be used as diagnosticmarkers, to monitor exposure to BMAA, and/or to identify diseaserelevant and functionally important proteins in which BMAA has beenincorporated in specific sequence locations. The proteins and peptidesdescribed herein can be utilized in a protein-cleavage-isotope dilutionworkflow to confirm the primary structure, and to accurately andprecisely quantify BMAA incorporated into peptides produced via chemicalor enzymatic digestion of specific proteins.

These proteins and peptides can also be employed in separation schemes,followed by intact mass spectrometry or peptide level detection andquantification through chemical or proteolytic digestion. These proteinsand peptides can also be used to produce antibodies, aptamers and/orother affinity reagents which can be utilized for other diagnostic toolsand applications. These proteins and peptides can also be used inbiophysical studies, providing a method for studying the effect of thisnon-protein amino acid incorporation in proteins.

By way of non-limiting illustration, examples of certain embodiments ofthe present disclosure are given below.

EXAMPLES Example 1: Preparation of ¹³C₄ ¹⁵N₂ β-N-Methylamino-L-Alanine

Preparation of Compound 1.

To a solution of ¹³C-¹⁵N labeled L-asparagine monohydrate (95.8 mg,0.614 mmol) in 10% aqueous Na₂CO₃ (1.6 mL) was added 1,4-dioxane (0.9mL) and the mixture was cooled to 0° C. Benzyl chloroformate (130 mg,0.737 mmol) was then added and the mixture was allowed to warm to rtovernight. The reaction mixture was poured into water (4.0 mL), and themixture was extracted with diethyl ether (×3). The aqueous layer wasthen acidified with an aqueous solution of 2N HCl (pH=2), and the whitesolid was filtered to afford 98.5 mg (59%) of the product: ¹H NMR (400MHz, CDCl₃) δ 7.37 (m, 5H), 5.10 (m, 2H), 4.25 (bd, 1H, J=132.1 Hz),2.81 (bd, 1H, J=44.1 Hz), 2.46 (bd, 1H, J=64.6 Hz); ESIMS m/z 273[M+H]⁺; HRMS m/z calculated for ¹³C₄C₈H₁₄ ¹⁵N₂O₅ [M+Na]⁺ 295.0870, found295.0867.

Preparation of Compound 2.

To a slurry of N²-benzyloxycarbonylasparagine (98.5 mg, 0.362 mmol) inethyl acetate (0.89 mL), acetonitrile (0.96 mL), and water (0.46 mL) wasadded iodosobenzene diacetate (0.166 g, 0.507 mmol) at 15° C. and themixture was stirred for 30 min at 15° C. The reaction mixture was thenallowed to warm to rt and stirred until completion (4 h). The mixturewas cooled to 5° C., and the product was collected, washed with ethylacetate, and dried in vacuo to afford 33.9 mg (39%) of the product as awhite solid: ¹H NMR (400 MHz, CDCl₃) δ 8.03 (brs, 1H), 7.85 (brs, 2H),7.40 (s, 5H), 5.07 (s, 2H), 4.30 (bd, 1H, J=139.2 Hz), 3.31 (bd, 1H,J=83.9 Hz), 3.31 (bd, 1H, J=83.9 Hz), 2.95 (bd, 1H, J=87.9 Hz); ESIMSm/z 244 [M+H]⁺; HRMS m/z calculated for ¹³C₃C₈H₁₄ ¹⁵N₂O₄ [M+H]⁺244.1068, found 244.1066.

Preparation of Compound 3.

To a suspension of compound 3 (33.9 mg, 0.139 mmol) in methanol (0.60mL) was added Et₃N (42.3 mg, 0.416 mmol) and benzaldehyde (29.6 mg,0.278 mmol) at rt, and the mixture was stirred for 30 min. The reactionmixture was cooled to 0° C., followed by the addition of NaBH₄ (16.0 mg,0.416 mmol). The mixture was then stirred for an additional 15 min at 0°C., and concentrated under reduced pressure. The residue was thendissolved in 0.1 M aqueous solution of NaOH, and extracted with diethylether (×3). The aqueous layer was then acidified with an aqueoussolution of 10% hydrochloric acid, and the resultant white precipitatewas filtered to afford 27.2 mg of the product. The white solid wasdissolved in methanol (0.27 mL), and a solution of 35% aqueous solutionof formaldehyde (18.2 μL, 0.244 mmol) was added. The reaction wasstirred for an additional 15 min, and cooled to 0° C. NaBH₄ (9.32 mg,0.244 mmol) was then added, and the mixture was stirred for 15 min. Uponcompletion, the mixture was concentrated under reduced pressure, and thecrude residue was dissolved in water, acidified (pH=6) with a 1 Maqueous solution of HCl, extracted with CHCl₃, dried (MgSO₄), andconcentrated under reduced pressure to afford the crude product. Thecrude product was triturated with diethyl ether to afford 28.3 mg (100%)of the product as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 7.18 (s,10H), 6.38 (d, 1H, J=92.5 Hz), 5.09 (d, 1H, J=16.5 Hz), 4.88 (d, 1H,J=15.4 Hz), 4.19 (d, 1H, J=83.9 Hz), 3.58 (d, 1H, J=13.2 Hz), 3.48 (d,1H, J=16.1 Hz), 3.01 (s, 1H), 2.67 (s, 1H), 2.12 (s, 3H); ESIMS m/z 348[M+H]⁺; HRMS m/z calculated for ¹³C₃C₁₆H₂₂ ¹⁵N₂O₄ [M+H]⁺ 348.1694, found348.1690.

Preparation of Compound ¹³C₄ ¹⁵N₂ β-N-Methylamino-L-Alanine.

To a degassed solution of compound 3 (28.3 mg, 0.0814 mmol) in methanol(0.8 mL) was added Pd/C (8.66 mg, 0.00813 mmol), and the mixture wasfurther degassed for an additional 5 min. The mixture was then saturatedwith H₂ gas and stirred under a H₂ atmosphere overnight. The Pd/C wasfiltered through Celite®, and washed with methanol. The filtrated wasconcentrated under reduced pressure and the crude product was trituratedwith diethyl ether to afford 7.7 mg (77%) of the product as a whitesolid: ESIMS m/z 146 [M+Na]⁺; HRMS m/z calculated for ¹³C₃CH₁₀ ¹⁵N₂O₂[M+H]⁺ 124.0856, found 124.0856. This final product was characterized byMS/MS to confirm the exact structure (FIG. 11).

A well-characterized, highly phosphorylated protein, Beta-Casein, wasutilized as a positive control. The intact protein was reacted withmethylamine under the same conditions for peptide synthesis, convertingphosphoserines to BMAA. Purified SOD1 from 3 patients with sporadic ALSand 3 healthy controls were washed on a 10 kDa FASP filter (Millipore)and concentrated to 50 μL. 20 μg of protein was hydrolyzed using 50 μLof 6N HCl and incubating at 110° C. for 18 hours. 5 μL of 60.9 mM SILBMAA was spiked into the samples post-hydrolysis. Samples were thendried and resuspended in 100 μL of 0.001% Zwittergent 3-16. Directinfusion ESI MS/MS of SIL BMAA confirmed the location of isotopeincorporation A ZipChip (908 Devices) capillary electrophoresis wasutilized to separate BMAA prior to electrospray ionization massspectrometry. Accurate intact mass and migration time of the SIL reagentwas used to identify BMAA.

The peptide sequence DGVADVSIEDSVISLSGDHCIIGR with highlighted (bold andunderlined) Serine phosphorylated, carbamidomethylated Cysteine and ¹³C₆¹⁵N₄ isotopically labeled Arginine was obtained from New EnglandPeptide. This sequence was validated by accurate intact mass (FIG. 2)and MS/MS (FIG. 3) which allowed for site specific confirmation ofphosphorylation. A solution of water, DMSO, EtOH solution in 2:2:1mixture containing 0.1M Barium Hydroxide and 1M Methylamine pH 12.5 wasused for derivatization of the SIL peptide. 5 μL (5 μg) of peptide wasadded to 20 μL of derivatization solution and incubated at 37° C. fortwo hours. The reaction was quenched with 2 μL of Acetic Acid. See FIG.1 for synthesis reference. The formation of BMAA at the phosphorylatedSerine residue was confirmed by accurate intact mass (FIG. 2) and MS/MS(FIG. 4). Further, the R.T of the BMAA peptide was distinctly differentfrom that of the phosphorylated peptide (FIG. 5), along with co-elutionof fragment ions to confirm the identity of this species. The conservedrelative abundance of the fragments belonging to the SIL BMAA peptideact as a standard for further confirmation.

Each sample was diluted 2-fold in a denaturing solution of 100 mM DTT(15.43 mg/mL) and 8 M urea. Then the samples were incubated at 56° C.for 30 minutes. After incubation enough alkylation solution, made from 1M iodoacetamide (184.96 mg/mL) and 8 M urea, was added to give eachsample a final iodoacetamide concentration of 200 mM. Then the sampleswere incubated at 37° C. for an hour. The appropriate amount for eachsample was pipetted into an Amicon Ultra-0.1 MWCO-filter unit (10 kDa,Millipore). These were centrifuged for 15 minutes at 14,000×g and 20°C.; when finished the eluent was discarded. The remaining volume wasdiluted with 400 uL of a digestion buffer made of 2 M urea (102.12mg/mL) and 10 mM CaCl_2 (1.11 mg/mL). The samples were centrifuged againfor 15 minutes at 14,000×g and 20° C. This was repeated two more times,making sure to discard the eluent after each run. After the finalcentrifugation the collection tubes were changed and 45 uL of modifiedporcine trypsin reconstituted in 2 M urea and 10 mM CaCl_2 was added andthe samples were incubated at 37° C. overnight. Then the samples werequenched with 50 uL of 1% formic acid (v/v) and 0.001% Zwittergent 3-16and centrifuged for 15 minutes at 14,000×g and 20° C. 400 uL of quenchbuffer was added to the retained volume and centrifuged again for 15minutes at 14,000×g and 20° C. Samples were frozen at −80° C. andlyophilized in a speedvac. Immediately prior to analysis samples werereconstituted in 90 uL of Zwittergent 3-16. A BMAA peptide dry aliquotof 5 ug was resuspended in 500 uL of Zwittergent. A 45 uL aliquot of theprotein digest was then spiked with 5 uL of BMAA peptide standard forLC-MS analysis.

LC-MS/MS by parallel reaction monitoring was used to isolate, fragmentand perform accurate mass measurements of our target endogenous BMAApeptide and SIL BMAA peptides. Direct inject column configuration on aThermo Easy nano-LC 1000 system coupled to a QExactive High Field massspectrometer was used. Analytical columns were made using 75 um×15 cmPicoFrit columns (New Objective, Woburn, Mass.) which were self-packedwith Kinetex C18 26-um particles (phenomenex, Torrance, Calif., USA).The samples were loaded with a 10 uL injection volume of mobile phase A(98% water, 2% acetonitrile, and 0.2% formic acid) with a max pressureof 500 Bar. A 45 minute run from 2% to 30% mobile phase B (98%acetonitrile, 2% water, and 0.2% formic acid) was performed at a flowrate of 300 nL/min. The analysis had the following parameters: a sprayvoltage of +1750.00, capillary temperature of 325° C., a S-lens RF levelof 65.00, a MS/MS resolving power of 15,000, a 1e6 AGC target, a 1,000ms fill time, a 2.5 m/z isolation window with an isolation offset of 1.0m/z, a fixed first mass of 125.0 m/z, and a 20, 30 stepped normalizedcollision energy. There was an inclusion list containing 846.4228 m/zand 843.0867 m/z.

Without the SIL BMAA peptide, correct identification of this peakbecomes very difficult as other mass conflicts are present within 5 ppmof the endogenous BMAA peptide as shown in FIG. 7 where the mostabundant peak is the same sequence does not co-elute with the SILpeptide. When examining the correct retention time, the intact massappears to be present within 1.2 ppm mass accuracy and the correctcharge state (+3) is also identified (FIG. 8). Moreover, this peakco-elutes with our SIL peptide as shown in FIG. 9. Fragment ionscorresponding to the endogenous peptide with BMAA at the expectedlocation could be identified at the same retention time (FIG. 10).

The compositions and methods of the appended claims are not limited inscope by the specific compositions and methods described herein, whichare intended as illustrations of a few aspects of the claims. Anycompositions and methods that are functionally equivalent are intendedto fall within the scope of the claims. Various modifications of thecompositions and methods in addition to those shown and described hereinare intended to fall within the scope of the appended claims. Further,while only certain representative compositions and method stepsdisclosed herein are specifically described, other combinations of thecompositions and method steps also are intended to fall within the scopeof the appended claims, even if not specifically recited. Thus, acombination of steps, elements, components, or constituents may beexplicitly mentioned herein or less, however, other combinations ofsteps, elements, components, and constituents are included, even thoughnot explicitly stated.

The term “comprising” and variations thereof as used herein is usedsynonymously with the term “including” and variations thereof and areopen, non-limiting terms. Although the terms “comprising” and“including” have been used herein to describe various embodiments, theterms “consisting essentially of” and “consisting of” can be used inplace of “comprising” and “including” to provide for more specificembodiments of the invention and are also disclosed. Other than wherenoted, all numbers expressing geometries, dimensions, and so forth usedin the specification and claims are to be understood at the very least,and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, to be construed in light of thenumber of significant digits and ordinary rounding approaches.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

What is claimed is:
 1. A composition comprising an isotopically labeledcompound defined by the formula below

wherein R represents hydrogen or an amine protecting group; and whereinat least two of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and^(h)O are isotopically labeled with a stable isotope.
 2. The compositionof claim 1, wherein at least three of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N,^(f)N, ^(g)O, and ^(h)O are isotopically labeled with a stable isotope.3. The composition of claim 1 or 2, wherein at least four of ^(a)C,^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O are isotopicallylabeled with a stable isotope.
 4. The composition of any of claims 1-3,wherein at least three of ^(a)C, ^(b)c, ^(c)C, ^(d)C, ^(e)N, and ^(f)Nare isotopically labeled with a stable isotope.
 5. The composition ofany of claims 1-4, wherein at least four of ^(a)C, ^(b)C, ^(c)C, ^(d)C,^(e)N, and ^(f)N are isotopically labeled with a stable isotope.
 6. Thecomposition of any of claims 1-5, wherein ^(a)C, ^(b)C, ^(c)C, ^(d)C,^(e)N, and ^(f)N are isotopically labeled with a stable isotope.
 7. Thecomposition of any of claims 1-6, wherein the isotopically labeledcompound is defined by the formula below

wherein R represents hydrogen or an amine protecting group; wherein the¹³C isotopic enrichment factor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is atleast 25; and wherein the ¹⁵N isotopic enrichment factor for ^(e)N and^(f)N is at least
 100. 8. The composition of claim 7, wherein the ¹³Cisotopic enrichment factor for ^(a)C, ^(b)C, ^(c)C, and ^(d)C is atleast 80 and the ¹⁵N isotopic enrichment factor for ^(e)N and ^(f)N isat least
 200. 9. The composition of any of claims 1-8, wherein R ishydrogen.
 10. The composition of any of claims 1-8, wherein R representsa 9-fluorenylmethyloxycarbonyl group.
 11. The composition of any ofclaims 1-10, wherein the isotopically labeled compound comprises atleast 0.5% by weight of the composition.
 12. A composition comprising apolypeptide, wherein the polypeptide includes one or more isotopicallylabeled β-N-methylamino-L-alanine (BMAA) residues, wherein each of theone or more isotopically labeled BMAA residues is isotopically labeledwith one or more stable isotopes.
 13. The composition of claim 12,wherein each of the one or more isotopically labeled BMAA residues isisotopically labeled with two or more stable isotopes.
 14. Thecomposition of claim 12 or 13, wherein each of the one or moreisotopically labeled BMAA residues is defined by the formula below

wherein the ¹³C isotopic enrichment factor for ^(d)C is at least 25; andwherein the ¹⁵N isotopic enrichment factor for N is at least
 100. 15.The composition of claim 14, wherein the ¹³C isotopic enrichment factorfor ^(d)C is at least 80 and the ¹⁵N isotopic enrichment factor for N isat least
 200. 16. The composition of claim 14 or 15, wherein the ¹³Cisotopic enrichment factor for ^(a)C, ^(b)C, and ^(c)C is at least 25.17. The composition of any of claims 14-16, wherein the ¹³C isotopicenrichment factor for ^(a)C, ^(b)C, and ^(c)C is at least
 80. 18. Thecomposition of any of claims 14-17, wherein the ¹⁵N isotopic enrichmentfactor for ^(e)N is at least
 100. 19. The composition of any of claims14-18, wherein the ¹⁵N isotopic enrichment factor for ^(e)N is at least200.
 20. The composition of any of claims 12-19, wherein the polypeptideincludes two or more isotopically labeled β-N-methylamino-L-alanine(BMAA) residues.
 21. The composition of claim 12 or 13, wherein thepolypeptide is defined by the formula below

wherein m is an integer from 0 to 300 and n is an integer from 0 to 300,with the proviso that at least one of m and n is not 0; the ¹³C isotopicenrichment factor for ^(d)C is at least 25; the ¹⁵N isotopic enrichmentfactor for ^(f)N is at least 100; and independently for each occurrencein the polypeptide, R₁ is H and R₂ is selected from one of the following

or R₁ and R₂, together with the atoms to which they are attached, form afive-membered heterocycle defined by the structure below


22. The composition of claim 21, wherein the ¹³C isotopic enrichmentfactor for ^(d)C is at least 80 and the ¹⁵N isotopic enrichment factorfor ^(f)N is at least
 200. 23. The composition of claim 21 or 22,wherein the ¹³C isotopic enrichment factor for ^(a)C, ^(b)C, and ^(c)Cis at least
 25. 24. The composition of any of claims 21-23, wherein the¹³C isotopic enrichment factor for ^(a)C, ^(b)C, and ^(c)C is at least80.
 25. The composition of any of claims 21-24, wherein the ¹⁵N isotopicenrichment factor for ^(e)N is at least
 100. 26. The composition of anyof claims 21-25, wherein the ¹⁵N isotopic enrichment factor for ^(e)N isat least
 200. 27. The composition of any of claims 21-26, wherein thesum of m and n is from 1 to
 50. 28. The composition of any of claims21-27, wherein the sum of m and n is from 1 to
 30. 29. The compositionof any of claims 12-28, wherein the polypeptide comprises at least 0.5%by weight of the composition.
 30. A method for quantifying the amount ofβ-N-methylamino-L-alanine (BMAA) in a sample comprising: (i) analyzing atest sample by mass spectrometry; (ii) spiking the test sample with adefined amount of an isotopically labeled compound defined by theformula below

wherein R represents hydrogen or an amine protecting group, and at leasttwo of ^(a)C, ^(b)C, ^(c)C, ^(d)C, ^(e)N, ^(f)N, ^(g)O, and ^(h)O areisotopically labeled with a stable isotope; to provide a BMAA-spikedsample; (iii) analyzing the BMAA-spiked sample by mass spectrometry; and(iv) determining the amount of BMAA in the test sample by isotopedilution analysis.
 31. The method of claim 30, wherein the methodfurther comprises preparing the test sample, the BMAA-spiked sample, ora combination thereof for analysis by mass spectrometry.
 32. The methodof claim 31, wherein the test sample, the BMAA-spiked sample, or acombination thereof are prepared for analysis by mass spectrometry by amethod comprising chemical reactions with flight enhancers, chemicalfragmentation, enzymatic digestion, purification, or a combinationthereof.
 33. The method of claim 32, wherein the test sample, theBMAA-spiked sample, or a combination thereof are prepared for analysisby enzymatic digestion.
 34. A method for incorporating anisotopically-labeled β-N-methylamino-L-alanine (BMAA) residue into aprotein or peptide, the method comprising: providing a protein orpeptide that includes a phosphoserine residue; reacting the protein orpeptide to convert the phosphoserine residue to an α,β-unsaturated aminoacid residue; and reacting the α,β-unsaturated amino acid residue withmethylamine to provide a BMAA residue, wherein the methylamine isisotopically enriched with one or more stable isotopes.
 35. The methodof claim 34, wherein the methylamine has a ¹³C isotopic enrichmentfactor of at least 25 and an ¹⁵N isotopic enrichment factor of at least100.
 36. The method of claim 35, wherein the methylamine has a ¹³Cisotopic enrichment factor of at least 80 and an ¹⁵N isotopic enrichmentfactor of at least 200.